This proposal seeks to understand the nature and degree to which human stem cells can form cells of the osteogenic lineage. The central hypothesis is that expression of osteogenic potential resides within a single population of multipotent stem cells that phenotypically differs depending upon its locale. We will test this hypothesis by examining three populations of cells reported to have osteogenic potential: bone marrow hematopoietic cells (focusing on CD34-negative cells), mesenchymal stem cells, and a recently described population of human bone precursor cells. The first goal of this proposal is to determine whether these populations of Osteopoietic Stem Cells (OSC) are related. This is accomplished by viral based marking studies and in vitro evaluation of the osteogenic potential of these cells. We have developed isolation and purification protocols that allow us to examine the function of osteoprogenitor cells, preosteoblasts, and osteoblast. The viral marking studies will utilize both lenti- and retroviral mediated gene transfer of GFP, taking care to deplete the three populations of more mature cells via lineage-depletion. Using our clonal osteoprogenitor cell assay, we take an alternative approach in which GFP-positive OPC from each population of virally marked cells are analyzed for integration by inverse-PCR. As well, a Power-Analysis of large numbers of marked OPC will be use to overcome problems of OSC frequency, integration efficiency, and signal-dilution. The second aim is to examine if the osteogenic fate of OSC populations can be enhanced. Such fate decisions are hypothesized to be modulated via manipulation of cell-intrinsic and/or cell-extrinsic mechanisms. Intrinsic mechanisms are investigated by modulation of bone related transcription factors (e.g., Fral, delta-fos B and others) and evaluation of their affect on osteogenic commitment and ex vivo bone formation. Extrinsic control of osteogenic fate determine focuses on the role of an accessory cell population, recently identified in this laboratory, as well as enforced expression of bone-active cytokines by CHO cells that function as "artificial Stromal cells." The final set of experiments focuses on the fate of OSC populations in vivo. These studies will determine the degree to which OSC can "home" to the osteogenic microenvironment in immuno-compromised Nude/NOD/SCID mice. As well, we will explore the capacity of these cells to commit to the osteogenic lineage and restore bone formation in osteoporotic NOD/SCID mice, as well as mice with skeletal defects.